Although leading HIV-1 vaccines now routinely elicit potent NAbs, 2 problems are 1) NAbs usually target strain- specific glycan holes, limiting breadth, 2) NAbs are inconsistent among vaccinees. Regarding the latter point, we have found that eliminating glycan head group clashes reveals consistent tier 2 NAbs in all vaccinated animals, suggesting that NAbs often fail to navigate glycans. In stark contrast, broad NAbs (bNAbs) from HIV- 1+ donors frequently contact glycans rather than avoid them. Therefore, we hypothesize that, by promoting NAb-glycan contacts, we might improve vaccine breadth & consistency. Here, we propose sequential heterologous prime-boost (SHPB) immunizations to try to elicit bNAbs to 2 conserved protein/glycan epitopes (V2 & fusion peptide; FP). Both sites accommodate multiple NAb binding modes, facilitating epitope focused approaches. In Aim 1, we will identify a panel of ?5 diverse, multi-V2 bNAb-sensitive, well-expressed trimers. Although high trimer expression is essential, such Envs are uncommon. 2 ways to obtain useful trimers will be: 1) to test various V2-sensitive special strains & 2) KI V2-sensitivity into high-expressing strains. Selected Envs will be modified to: 1) Plug glycan holes, 2) KI the common FP variant 1 (FP8var1) sequence/KO the N611 glycan (regulates FP exposure). In Aim 2, we will try to improve vaccine NAbs, using virus-like particles (VLPs) expressing trimers from Aim 1. CH01 HC KI BL6 mice express the UCA of the CH01 NAb heavy chain (HC) amid a mouse HC repertoire that can combine with diverse mouse LCs. These mice can potentially generate NAbs to additional targets like FP. Since early events shape NAb responses, following initial KLH-FP8v1 priming a variety of concepts will be tested in 3 subsequent VLP shots, including glycosidase-digested VLPs to minimize clashes or immunogenic foreign glycans to promote early NAb-glycan contacts. Later VLP shots will be kept consistent to expand NAbs arising from earlier shots. 5 mice will be sacrificed at an intermediate timepoint & the rest after the final shot, allowing us to study NAb ontogeny. The best priming regimen will be determined primarily from neutralization kinetics, potency, breadth & consistency, & from molecular genetics criteria. This regimen will be re-tested in mice where CH01 precursors are reduced to physiologic frequency by adoptive transfer & in CH01 HC x Balb/c F1 mice that also exhibit reduced CH01 HC frequency & a more robust genetic background to potentiate NAb development. Having identified effective priming shots, assembled SHPB regimens will be tested in the same F1 mice to try to improve breadth. Variables will include overlapping or non-overlapping sequential shots, increasing strain diversity, & increasing epitope stringency. Finally, we will test leading regimens in Trianni mice expressing polyclonal human IgG. Serum neutralization of vaccine & non-vaccine strains will be monitored. MAbs will be rescued using VLP probes & analyzed for SHM, V gene use, & H/L pairings. NAb titers, breadth, effects of glycan changes, specificity, glycan array activity & ontogeny will be investigated. Overall, we hope to advance HIV vaccines by identifying regimens that induce glycan-dependent, V2 & FP-targeted NAb breadth.